In vitro generation of hematopoietic cells and especially hematopoietic stem cells (HSCs) from human pluripotent stem cells (PSCs) are subject to intensive research in recent decades, as these cells ...hold great potential for regenerative medicine and autologous cell replacement therapies. Despite many attempts, in vitro, de novo generation of bona fide HSCs remains challenging, and we are still far away from their clinical use, due to insufficient functionality and quantity of the produced HSCs. The challenges of generating PSC-derived HSCs are already apparent in early stages of hemato-endothelial specification with the limitation of recapitulating complex, dynamic processes of embryonic hematopoietic ontogeny in vitro. Further, these current shortcomings imply the incompleteness of our understanding of human ontogenetic processes from embryonic mesoderm over an intermediate, specialized hemogenic endothelium (HE) to their immediate progeny, the HSCs. In this review, we examine the recent investigations of hemato-endothelial ontogeny and recently reported progress for the conversion of PSCs and other promising somatic cell types towards HSCs with the focus on the crucial and inevitable role of the HE to achieve the long-standing goal—to generate therapeutically applicable PSC-derived HSCs in vitro.
The increasing number of severe infections with multi-drug-resistant pathogens worldwide highlights the need for alternative treatment options. Given the pivotal role of phagocytes and especially ...alveolar macrophages in pulmonary immunity, we introduce a new, cell-based treatment strategy to target bacterial airway infections. Here we show that the mass production of therapeutic phagocytes from induced pluripotent stem cells (iPSC) in industry-compatible, stirred-tank bioreactors is feasible. Bioreactor-derived iPSC-macrophages (iPSC-Mac) represent a highly pure population of CD45
CD11b
CD14
CD163
cells, and share important phenotypic, functional and transcriptional hallmarks with professional phagocytes, however with a distinct transcriptome signature similar to primitive macrophages. Most importantly, bioreactor-derived iPSC-Mac rescue mice from Pseudomonas aeruginosa-mediated acute infections of the lower respiratory tract within 4-8 h post intra-pulmonary transplantation and reduce bacterial load. Generation of specific immune-cells from iPSC-sources in scalable stirred-tank bioreactors can extend the field of immunotherapy towards bacterial infections, and may allow for further innovative cell-based treatment strategies.
The broad success of adoptive immunotherapy to treat human cancer has resulted in a paradigm shift in modern medicine. Modification of autologous and allogenic immune cells with chimeric antigen ...receptors (CAR) designed to target specific antigens on tumor cells has led to production of CAR T and CAR NK cell therapies, which are ever more commonly introduced into cancer patient treatment protocols. While allogenic T cells may offer advantages such as improved anti-tumor activity, they also carry the risk of adverse reactions like graft-versus-host disease. This risk can be mitigated by use of autologous immune cells, however, the time needed for T and/or NK cell isolation, modification and expansion may be too long for some patients. Thus, there is an urgent need for strategies to robustly produce "off-the-shelf" CAR T and CAR NK cells, which could be used as a bridging therapy between cancer diagnosis or relapse and allogeneic transplantation. Advances in genome modification technologies have accelerated the generation of designer cell therapy products, including development of "off-the-shelf" CAR T cells for cancer immunotherapy. The feasibility and safety of such approaches is currently tested in clinical trials. This review will describe cell sources for CAR-based therapies, provide background of current genome editing techniques and the applicability of these approaches for generation of universal "off-the-shelf" CAR T and NK cell therapeutics.
Genetic engineering T cells to create clinically applied chimeric antigen receptor (CAR) T cells has led to improved patient outcomes for some forms of hematopoietic malignancies. While this has ...inspired the biomedical community to develop similar strategies to treat solid tumor patients, challenges such as the immunosuppressive character of the tumor microenvironment, CAR-T cell persistence and trafficking to the tumor seem to limit CAR-T cell efficacy in solid cancers. This review provides an overview of mechanisms that tumors exploit to evade eradication by CAR-T cells as well as emerging approaches that incorporate genetic engineering technologies to improve CAR-T cell activity against solid tumors.
The stable retroviral integration of therapeutic transgene cassettes into patients' hematopoietic stem and progenitor cells enables gene therapy for various disorders of the blood and immune ...systems.1,2,3,4 However, adverse events related to insertional mutagenesis were observed in the first clinical gene therapy trials.5,6 These adverse events were caused mainly by the insertional activation of proto-oncogenes, such as LMO2 and MDS/EVI1, which occurred as a consequence of the retroviral integration preferences and the strength of the enhancer of the first-generation long terminal repeat (LTR)-driven gammaretroviral vectors. Two concepts have been proposed to achieve a "safer" integration profile.
Gene therapy can be used to restore cell function in monogenic disorders or to endow cells with new capabilities, such as improved killing of cancer cells, expression of suicide genes for controlled ...elimination of cell populations, or protection against chemotherapy or viral infection. While gene therapies were originally most often used to treat monogenic diseases and to improve hematopoietic stem cell transplantation outcome, the advent of genetically modified immune cell therapies, such as chimeric antigen receptor modified T cells, has contributed to the increased numbers of patients treated with gene and cell therapies. The advancement of gene therapy with integrating retroviral vectors continues to depend upon world-wide efforts. As the topic of this special issue is "Spotlight on Germany," the goal of this review is to provide an overview of contributions to this field made by German clinical and research institutions. Research groups in Germany made, and continue to make, important contributions to the development of gene therapy, including design of vectors and transduction protocols for improved cell modification, methods to assess gene therapy vector efficacy and safety (e.g., clonal imbalance, insertion sites), as well as in the design and conduction of clinical gene therapy trials.
Abstract
Genome editing with the CRISPR-Cas9 system has enabled unprecedented efficacy for reverse genetics and gene correction approaches. While off-target effects have been successfully tackled, ...the effort to eliminate variability in sgRNA efficacies-which affect experimental sensitivity-is in its infancy. To address this issue, studies have analyzed the molecular features of highly active sgRNAs, but independent cross-validation is lacking. Utilizing fluorescent reporter knock-out assays with verification at selected endogenous loci, we experimentally quantified the target efficacies of 430 sgRNAs. Based on this dataset we tested the predictive value of five recently-established prediction algorithms. Our analysis revealed a moderate correlation (r = 0.04 to r = 0.20) between the predicted and measured activity of the sgRNAs, and modest concordance between the different algorithms. We uncovered a strong PAM-distal GC-content-dependent activity, which enabled the exclusion of inactive sgRNAs. By deriving nine additional predictive features we generated a linear model-based discrete system for the efficient selection (r = 0.4) of effective sgRNAs (CRISPRater). We proved our algorithms' efficacy on small and large external datasets, and provide a versatile combined on- and off-target sgRNA scanning platform. Altogether, our study highlights current issues and efforts in sgRNA efficacy prediction, and provides an easily-applicable discrete system for selecting efficient sgRNAs.
Gene therapy on the move Kaufmann, Kerstin B.; Büning, Hildegard; Galy, Anne ...
EMBO molecular medicine,
November 2013, Volume:
5, Issue:
11
Journal Article
Peer reviewed
Open access
The first gene therapy clinical trials were initiated more than two decades ago. In the early days, gene therapy shared the fate of many experimental medicine approaches and was impeded by the ...occurrence of severe side effects in a few treated patients. The understanding of the molecular and cellular mechanisms leading to treatment‐ and/or vector‐associated setbacks has resulted in the development of highly sophisticated gene transfer tools with improved safety and therapeutic efficacy. Employing these advanced tools, a series of Phase I/II trials were started in the past few years with excellent clinical results and no side effects reported so far. Moreover, highly efficient gene targeting strategies and site‐directed gene editing technologies have been developed and applied clinically. With more than 1900 clinical trials to date, gene therapy has moved from a vision to clinical reality. This review focuses on the application of gene therapy for the correction of inherited diseases, the limitations and drawbacks encountered in some of the early clinical trials and the revival of gene therapy as a powerful treatment option for the correction of monogenic disorders.
A timely review on the application of gene therapy for the correction of inherited diseases, including a description of the drawbacks from the early clinical trials, how the inherent risks are being successfully addressed and future perspectives.
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